A precursor cell (stem, germinal, undifferentiated, or primitive) is a type of cell with self-renewal capacity for a significant time period and, above all, with the capacity of either partial or terminal differentiation into other types of more specialized cells.
Despite the enormous differentiation potential of embryonic precursor cells, their utilization for research or/and therapy is controversial and has raised serious ethical and safety issues. Thus, research in this area has been focusing on the identification and evaluation of alternative non-embryonic stem cells, such as those obtained from bone marrow, periostium, trabecular bone, adipose tissue, sinovial region, skeletal muscle, deciduous and definitive teeth pulp, and olfactory mucosa (Barry and Murphy, 2004; Roisen et al., 2001). It has already been demonstrated that cells isolated from these tissues have the capacity of differentiation inter alia into chondrocytes, adipocytes, osteoblasts, myoblasts, cardiomyocytes, astrocytes and tenocytes, both in vitro and in vivo (Carvalhal et al., 2007; Majumdar et al., 1998 e Pittenger et al., 1999). Such precursor cells, isolated from non-embryonic sources, and capable of differentiating into non-haematopoietic specialized cells, derived from the three germ layers (endoderm, mesoderm, and ectoderm), are denominated mesenchymal stem cells.
The major limitations to the utilization of mesenchymal precursor cells arise during clinical practice, namely during cell harvesting. Collection of mesenchymal cells invariably involves invasive methodologies to the donor, such as surgical procedures that (like collection of stem cells from bone marrow, for example) might even involve general anaesthesia. Furthermore, because mesenchymal stem cells are rare, the final number of cells obtained is generally low.
As an alternative, the umbilical cord tissues have been described as possible sources for adult precursor cells (Romanov et al., 2003). The umbilical cord blood, for example, is known to be a rich source for precursor cells but mainly of haematopoietic nature (blood lineage). Since mesenchymal stem cells are present in umbilical cord blood in limited numbers, attempts to isolate these cells from this tissue have resulted in some frustration, and even the most successful attempts, using very high amounts of blood, have not surpassed the 60% success rate, relative to the total number of tissue samples processed. And in the end, doubts still persisted about the origin of the isolated cells; that is, would the origin of cells have really been blood, or other foetal tissue (Chul-Wan et al., 2003; Bieback et al., 2004).
Other reports describe isolation of mesenchymal stem cells from other umbilical cord constituent tissues, considerably richer in mesenchymal nature than umbilical blood. Some examples of these procedures are based on umbilical cord matrix, also known by Wharton's jelly (Purchio et al., 1998; Mitchell et al., 2003; Davies et al., 2004; Wang et al., 2006); umbilical cord vein (Romanov et al., 2003; Auger et al., 2005), arterial tissues (Kadner et al., 2004); or other lining tissues, such as the amniotic membrane (Phan et al., 2004).
A detailed analysis reveals that the protocols described are, in one way or the other, restrictive in terms of the nature of the cells obtained, or rather vague in terms of success rate and efficiency in the number of cells isolated. In fact, the restrictive nature of these protocols invariably resulted in loss of phenotypic diversity of the cell populations isolated, mainly due to unnecessary focus on specific tissues or geographic locations within the umbilical cord structure. Furthermore, uncertainly invariably remained about the actual number of stem cells that could be obtained in the end.
Thus, for example, the Cell Research Corporation protocol that is based solely on the amniotic membrane as source of mesenchymal stem cells, originates cells already pre-disposed to the endothelial lineage (Phan et al., 2004).
Additionally, none of the methods described so far has demonstrated efficacy in terms of number of successful tissue samples processed in order to be reliable enough so as to be applied in cell therapy protocols. In other words, although the success rate for mesenchymal cell isolation from umbilical matrix is higher than from umbilical cord blood, or even bone marrow, there is no method up to now that guarantees 100% success rate for isolation, in terms of number of tissue samples processed, so that the final result is robust enough for cell therapy applications (Deryl e Weiss, 2008).
Furthermore, the introduction of unnecessary steps of structural manipulation, such as extraction of umbilical vessels (Purchio et al., 1998; Mitchell et al., 2003; Davies et al., 2004; Wang et al., 2006), or mechanical maceration (Seyda et al., 2006), makes existing protocols hard to standardize and reproduce, never assuring enough cell numbers for cell therapy application.
Furthermore, excessive tissue manipulation induces cell differentiation which is undesirable if one wants to maintain precursor cell phenotype (Gardner et al., 2000; Claes et al., 2002; Cullinane et al., 2003).
Also showing limitations are the protocols based on the umbilical vessels themselves. These protocols involve complex extractions of the arteries or the umbilical vein and limit the differentiation potential of mesenchymal stem cells to the sub-endothelial and endothelial lineages (Romanov et al., 2003; Auger et al., 2005; Kadner et al., 2004; Sarugaser et al., 2005).
Finally, no less complex, are the protocols that base themselves on the Wharton's jelly (WJ) as source for mesenchymal stem cells. These reports are not consistent, also contributing to the lack of definition and criteria of the applied methodologies. Thus, while Purchio et al., 1998; Mitchell et al., 2003; and Wang et al., 2006, perform a complex and hardly reproducible initial vessel extraction, processing the remaining tissue for cell isolation, Davies et al., 2004, also remove the umbilical vessels but, instead of using the remaining tissue, they process the tissue still coupled around the vessels for cell isolation, discarding the first. Nonetheless, all authors are unanimous in affirming that their protocols are based on WJ exclusively (Purchio et al., 1998; Mitchell et al., 2003; Davies et al., 2004; Wang et al., 2006). The discrepancy between these approaches is unjustified and the excess tissue manipulation of the existing tissues in either of these two protocols undermines desirable effects on precursor phenotype maintenance, and consequently endangers the utilization of the isolated precursor cells in isolation and cryopreservation services for the population in general.
The state-of-the-art is clearly missing a method based on a simple, robust, and defined protocol, so that it can be reproduced with guarantees of efficacy and efficiency. Once mesenchymal stem cells become applicable in cell therapy, it is necessary to assure to the patient that the method used for cell isolation will provide both the necessary quality and quantity of the therapeutic agent. Given the lack of guarantees presented by the protocols so far described in the state-of-the-art, it is foreseen that the present invention will suppress the need for a method with the above characteristics.